Als D et al. Global trends in typhoidal salmonellosis: A systematic review. (Submitted 2017). No Abstract Available.

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1 References for Typhoid Vaccines: WHO Position Paper, March 2018 Acharya VI et al. Prevention of typhoid fever in Nepal with the Vi capsular polysaccharide of Salmonella typhi. A preliminary report. N Engl J Med. 1987;317: We conducted a pilot study followed by a large clinical trial in Nepal of the use of the capsular polysaccharide of Salmonella typhi (Vi) as a vaccine to prevent typhoid fever. In the pilot study, involving 274 Nepalese, there were no significant side effects of the Vi vaccine; about 75 percent responded with a rise in serum antibodies of fourfold or more. In the clinical trial, residents of five villages were given intramuscular injections of either Vi or, as a control, pneumococcus vaccine dispensed in coded, randomly arranged, single-dose syringes. There were 6907 participants, of whom 6438 were members of the target population (5 to 44 years of age); each was visited every two days. Those with temperatures of 37.8 degrees C or higher for three consecutive days were examined and asked to give blood for culture. Typhoid was diagnosed as either blood culture-positive or clinically suspected on the basis of bradycardia, splenomegaly, and fever, with a negative blood culture. Seventeen months after vaccination, the codes were broken for the 71 patients meeting the criteria for either culture-positive or clinically suspected typhoid. The attack rate of typhoid was 16.2 per 1000 among the controls and 4.1 per 1000 among those immunized with Vi (P less than ). The efficacy of Vi was 72 percent in the culture-positive cases, 80 percent in the clinically suspected cases, and 75 percent in the two groups combined. These data provide evidence that Vi antibodies confer protection against typhoid. Surveillance continues to determine the duration of Vi-induced immunity. Als D et al. Global trends in typhoidal salmonellosis: A systematic review. (Submitted 2017). No Abstract Available. Antillon M et al. Cost-effectiveness analysis of typhoid conjugate vaccines in five endemic low- and middle-income settings. Vaccine. 2017;35: Background: Typhoid fever remains endemic in low- and middle-income countries. Programmatic use of existing vaccines is limited, but upcoming typhoid conjugate vaccines (TCVs) could warrant wider use. We evaluated the cost-effectiveness of five TCV delivery strategies in three urban areas (Delhi and Kolkata, India and Nairobi, Kenya) and two rural settings (Lwak, Kenya and Dong Thap, Vietnam) with varying incidence. Methods and findings: We evaluated routine infant vaccination with and without catch-up campaigns among older individuals. We used a dynamic model of typhoid transmission to simulate cases, hospitalizations, deaths, disability-adjusted life-years (DALY) lost, treatment and intervention costs. We estimated cost-effectiveness (in terms of cost in international dollars (I$) per DALY averted) from the healthcare payer perspective, and assessed how it was influenced by uncertain model parameters. Compared to no vaccination, routine infant vaccination at I$1/dose was cost-saving in Delhi and Dong Thap, very cost-effective in Kolkata and Nairobi, and cost-effective in Lwak according to World Health Organization thresholds. However, routine vaccination was not the optimal strategy compared to strategies that included a catch-up campaign, which yielded the highest probability of being cost-saving in Delhi and Dong Thap and were most likely to provide a return on investment above a willingness-to-pay threshold of I$1440 in Kolkata, I$2300 in Nairobi, and I$5360 in Lwak. Vaccine price impacted the optimal strategy, and the number of doses required and rate of hospitalization were the primary sources of uncertainty. Conclusion: Routine vaccination with TCV would be cost-effective in most settings, and additional one-time catch-up campaigns would also be economically justified. Arjyal A et al. Gatifloxcain versus ceftriaxone for uncomplicated enteric fever in Nepal: an open-label, two-centre, randomised controlled trial. Lancet Infect Dis. 2016;16:

2 Background: Because treatment with third-generation cephalosporins is associated with slow clinical improvement and high relapse burden for enteric fever, whereas the fluoroquinolone gatifloxacin is associated with rapid fever clearance and low relapse burden, we postulated that gatifloxacin would be superior to the cephalosporin ceftriaxone in treating enteric fever. Methods : We did an open-label, randomised, controlled, superiority trial at two hospitals in the Kathmandu valley, Nepal. Eligible participants were children (aged 2 13 years) and adult (aged years) with criteria for suspected enteric fever (body temperature 38 0 C for 4 days without a focus of infection). We randomly assigned eligible patients (1:1) without stratification to 7 days of either oral gatifloxacin (10 mg/kg per day) or intravenous ceftriaxone (60 mg/kg up to 2 g per day for patients aged 2 13 years, or 2 g per day for patients aged 14 years). The randomisation list was computer-generated using blocks of four and six. The primary outcome was a composite of treatment failure, defined as the occurrence of at least one of the following: fever clearance time of more than 7 days after treatment initiation; the need for rescue treatment on day 8; microbiological failure (ie, blood cultures positive for Salmonella enterica serotype Typhi, or Paratyphi A, B, or C) on day 8; or relapse or disease-related complications within 28 days of treatment initiation. We did the analyses in the modified intention-to-treat population, and subpopulations with either confirmed blood-culture positivity, or blood-culture negativity. The trial was powered to detect an increase of 20% in the risk of failure. This trial was registered at ClinicalTrials.gov, number NCT , and is now closed. Findings: Between Sept 18, 2011, and July 14, 2014, we screened 725 patients for eligibility. On July 14, 2014, the trial was stopped early by the data safety and monitoring board because S Typhi strains with high-level resistance to ciprofloxacin and gatifloxacin had emerged. At this point, 239 were in the modified intention-to-treat population (120 assigned to gatifloxacin, 119 to ceftriaxone). 18 (15%) patients who received gatifloxacin had treatment failure, compared with 19 (16%) who received ceftriaxone (hazard ratio [HR] 1 04 [95% CI ]; p=0 91). In the culture-confirmed population, 16 (26%) of 62 patients who received gatifloxacin failed treatment, compared with four (7%) of 54 who received ceftriaxone (HR 0 24 [95% CI ]; p=0 01). Treatment failure was associated with the emergence of S Typhi exhibiting resistance against fluoroquinolones, requiring the trial to be stopped. By contrast, in patients with a negative blood culture, only two (3%) of 58 who received gatifloxacin failed treatment versus 15 (23%) of 65 who received ceftriaxone (HR 7 50 [95% CI ]; p=0 01). A similar number of non-serious adverse events occurred in each treatment group, and no serious events were reported. Interpretation: Our results suggest that fluoroquinolones should no longer be used for treatment of enteric fever in Nepal. Additionally, under our study conditions, ceftriaxone was suboptimum in a high proportion of patients with culture-negative enteric fever. Since antimicrobials, specifically fluoroquinolones, are one of the only routinely used control measures for enteric fever, the assessment of novel diagnostics, new treatment options, and use of existing vaccines and development of next-generation vaccines are now a high priority. Azmatullah A et al. Systematic review of the global epidemiology, clinical and laboratory profile of enteric fever. J Glob Health. 2015;5: Children suffer the highest burden of enteric fever among populations in South Asian countries. The clinical features are non specific, vary in populations, and are often difficult to distinguish clinically from other febrile illnesses, leading to delayed or inappropriate diagnosis and treatment. We undertook a systematic review to assess the clinical profile and laboratory features of enteric fever across age groups, economic regions, level of care and antibiotic susceptibility patterns. Baker S et al. Combined high-resolution genotyping and geospatial analysis reveals modes of endemic urban typhoid fever transmission. Open Biol. 2011;1:

3 Typhoid is a systemic infection caused by Salmonella Typhi and Salmonella Paratyphi A, humanrestricted bacteria that are transmitted faeco-orally. Salmonella Typhi and S. Paratyphi A are clonal, and their limited genetic diversity has precluded the identification of long-term transmission networks in areas with a high disease burden. To improve our understanding of typhoid transmission we have taken a novel approach, performing a longitudinal spatial case control study for typhoid in Nepal, combining single-nucleotide polymorphism genotyping and case localization via global positioning. We show extensive clustering of typhoid occurring independent of population size and density. For the first time, we demonstrate an extensive range of genotypes existing within typhoid clusters, and even within individual households, including some resulting from clonal expansion. Furthermore, although the data provide evidence for direct human-to-human transmission, we demonstrate an overwhelming contribution of indirect transmission, potentially via contaminated water. Consistent with this, we detected S. Typhi and S. Paratyphi A in water supplies and found that typhoid was spatially associated with public water sources and low elevation. These findings have implications for typhoid-control strategies, and our innovative approach may be applied to other diseases caused by other monophyletic or emerging pathogens. Bhutta ZA. Impact of age and drug resistance on mortality in typhoid fever. Arch Dis Child. 1996;75: The risk factors for mortality were analysed in a consecutive group of 1158 children presenting to the Aga Khan University Medical Center, Karachi, with multidrug resistant typhoid fever that had been proved on culture. There were 19 deaths, representing an overall case fatality rate of 1.6%. Multidrug resistant typhoid was associated with a more severe clinical illness and higher rates of toxicity, hepatomegaly, hypotensive shock, and death. Irrespective of drug resistance status, typhoid fever was found to be a more severe illness in young infants with significantly higher rates of diarrhoea, hypotensive shock, and mortality. Univariate analysis of admission characteristics associated with increased risk for mortality revealed significant association with younger age (p < 0.05), hypotensive shock or hypothermia (p < 0.001), obtundation (p < 0.001), seizures (p < 0.05), anaemia at admission (p < 0.005), and leucocytosis (p < 0.001). Logistic regression analysis of risk factors for mortality showed persistent association of hypothermia, toxicity, and anaemia with mortality. The data provides evidence that multidrug resistant typhoid in childhood is associated with increased risk of mortality, especially in infancy and closer attention to several risk factors for increased morbidity and case fatality rates may lead to improved outcome of treatment. Breiman RF et al. Population-based incidence of typhoid fever in an urban informal settlement, Nairobi, Kenya: implications for typhoid vaccine use in Africa. PLoS One. 2012;7:e Background: High rates of typhoid fever in children in urban settings in Asia have led to focus on childhood immunization in Asian cities, but not in Africa, where data, mostly from rural areas, have shown low disease incidence. We set out to compare incidence of typhoid fever in a densely populated urban slum and a rural community in Kenya, hypothesizing higher rates in the urban area, given crowding and suboptimal access to safe water, sanitation and hygiene. Methods: During , we conducted population-based surveillance in Kibera, an urban informal settlement in Nairobi, and in Lwak, a rural area in western Kenya. Participants had free access to study clinics; field workers visited their homes biweekly to collect information about acute illnesses. In clinic, blood cultures were processed from patients with fever or pneumonia. Crude and adjusted incidence rates were calculated. Results: In the urban site, the overall crude incidence of Salmonella enterica serovar Typhi (S. Typhi) bacteremia was 247 cases per 100,000 person-years of observation (pyo) with highest rates in children 5-9 years old (596 per 100,000 pyo) and 2-4 years old (521 per 100,000 pyo). Crude overall incidence in Lwak was 29 cases per 100,000 pyo with low rates in children 2-4 and 5-9 years old (28 and 18 cases per 100,000 pyo,

4 respectively). Adjusted incidence rates were highest in 2-4 year old urban children (2,243 per 100,000 pyo) which were >15-fold higher than rates in the rural site for the same age group. Nearly 75% of S. Typhi isolates were multi-drug resistant. Conclusions: This systematic urban slum and rural comparison showed dramatically higher typhoid incidence among urban children <10 years old with rates similar to those from Asian urban slums. The findings have potential policy implications for use of typhoid vaccines in increasingly urban Africa. Britto C et al. An appraisal of the clinical features of pediatric enteric fever: systematic review and meta-analysis of the age-stratified disease occurrence. Clin Infect Dis. 2017;64: Children bear a substantial proportion of the enteric fever disease burden in endemic areas. Controversy persists regarding which age groups are most affected, leading to uncertainty about optimal intervention strategies. We performed a systematic review and meta-analysis of studies in Asia and Africa to compare the relative proportion of children with enteric fever in the age groups <5 years, 5 9 years, and years. Overall, studies conducted in Africa showed a relatively smaller occurrence of disease in the youngest age group, whereas in Asia the picture was more mixed with a very large degree of heterogeneity in estimates. The clinical features of enteric fever reviewed here differ between younger and older children and adults, likely leading to further uncertainty over disease burden. It is evident from our review that preschool children and infants also contribute a significant proportion of disease burden but have not been adequately targeted via vaccination programs, which have been focusing primarily on school-based vaccination campaigns. Carias C. et al. Economic evaluation of typhoid vaccination in a prolonged typhoid outbreak setting: the case of Kasese district in Uganda. Vaccine 2015;33: Background:Vaccination has been increasingly promoted to help control epidemic and endemic typhoid fever in high-incidence areas. Despite growing recognition that typhoid incidence in some areas of sub- Saharan Africa is similar to high-incidence areas of Asia, no large-scale typhoid vaccination campaigns have been conducted there. We performed an economic evaluation of a hypothetical one-time, fixedpost typhoid vaccination campaign in Kasese, a rural district in Uganda where a large, multi-year outbreak of typhoid fever has been reported. Methods: We used medical cost and epidemiological data retrieved on-site and campaign costs from previous fixed-post vaccination campaigns in Kasese to account for costs from a public sector health care delivery perspective. We calculated program costs and averted disability-adjusted life years (DALYs) and medical costs as a result of vaccination, to calculate the cost of the intervention per DALY and case averted. Results: Over the 3 years of projected vaccine efficacy, a one-time vaccination campaign was estimated to avert 1768 (90%CI: ) typhoid fever cases per year and a total of 3868 (90%CI: ) DALYs over the duration of the immunity conferred by the vaccine. The cost of the intervention per DALY averted was US$ 484 (90%CI: ) and per case averted US$ 341 (90%CI: ). Conclusion: We estimated the vaccination campaign in this setting to be highly cost-effective, according to WHO's cost-effective guidelines. Results may be applicable to other African settings with similar high disease incidence estimates. Carles G et al. Typhoid fever and pregnancy. J Gynecol Obstet Biol Reprod. 2002;31: Typhoid fever is rare in Europe, but well-recognized endemic disease in tropical zones. We report our findings in a series of 25 cases of typhoid fever during pregnancy observed in French Guiana and reviewed the literature on clinical signs, diagnosis and treatment. Salmonellea typhi causes septicemia of digestive origin that can cross the placenta resulting in chorioamniotitis. Maternal-fetal infection with S. typhi can lead to miscarriage, fetal death, neonatal infection, as well as diverse maternal complications. In order to avoid maternal complications and possible fetal transmission, treatment with ceftriaxone should be initiated as early as possible.

5 Cook J. et al. The cost-effectiveness of typhoid Vi vaccination programs: calculations for four urban sites in four Asian countries. Vaccine. 2008;26: The burden of typhoid fever remains high in impoverished settings, and increasing antibiotic resistance is making treatment costly. One strategy for reducing the typhoid morbidity and mortality is vaccination with the Vi polysaccharide vaccine. We use a wealth of new economic and epidemiological data to evaluate the cost-effectiveness of Vi vaccination against typhoid in sites in four Asian cities: Kolkata (India), Karachi (Pakistan), North Jakarta (Indonesia), and Hue (Vietnam). We report results from both a societal as well as a public sector financial perspective. Baseline disease burden estimates in the four areas are: 750 cases per year in two Kolkata neighborhoods (pop 185,000); 84 cases per year in the city of Hue (pop 280,000); 298 cases per year in two sub-districts in North Jakarta (pop 161,000), and 538 cases per year in three squatter settlements in Karachi (pop 102,000). We estimate that a vaccination program targeting all children (2 14.9) would prevent 456, 158, and 258 typhoid cases (and 4.6, 1.6, and 2.6 deaths), and avert 126, 44, and 72 disability-adjusted life years (DALYs) over 3 years in Kolkata, North Jakarta and Karachi, respectively. The net social costs would be US$160 and US$549, per DALY averted in Kolkata and North Jakarta, respectively. These programs, along with a similar program in Karachi, would be considered very cost-effective (e.g. costs per DALY averted less than per capita gross national income (GNI)) under a wide range of assumptions. Community-based vaccination programs that also target adults in Kolkata and Jakarta are less cost-effective because incidence is lower in adults than children, but are also likely to be very cost-effective. A program targeting school-aged children in Hue, Vietnam would prevent 21 cases, avert 6 DALYs, and not be cost-effective (US$3779 per DALY averted) because of the low typhoid incidence there. Crump JA et al. Epidemiology, clinical presentation, laboratory diagnosis, antimicrobial resistance and antimicrobial management of invasive Salmonella infections. Clin Microbiol Rev. 2015;28: Summary> Salmonella enterica infections are common causes of bloodstream infection in low-resource areas, where they may be difficult to distinguish from other febrile illnesses and may be associated with a high case fatality ratio. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis. Antimicrobial resistance has emerged in Salmonella enterica, initially to the traditional first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. Decreased fluoroquinolone susceptibility and then fluoroquinolone resistance have developed in association with chromosomal mutations in the quinolone resistance-determining region of genes encoding DNA gyrase and topoisomerase IV and also by plasmid-mediated resistance mechanisms. Resistance to extendedspectrum cephalosporins has occurred more often in nontyphoidal than in typhoidal Salmonella strains. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug in areas where fluoroquinolone resistance is common. In 2013, CLSI lowered the ciprofloxacin susceptibility breakpoints to account for accumulating clinical, microbiologic, and pharmacokinetic-pharmacodynamic data suggesting that revision was needed for contemporary invasive Salmonella infections. Newly established CLSI guidelines for azithromycin and Salmonella enterica serovar Typhi were published in CLSI document M100 in Darton TC et al. Using a Human Challenge Model of Infection to Measure Vaccine Efficacy: A Randomised, Controlled Trial Comparing the Typhoid Vaccines M01ZH09 with Placebo and Ty21a. PLoS Negl Trop Dis. 2016;10:e Typhoid persists as a major cause of global morbidity. While several licensed vaccines to prevent typhoid are available, they are of only moderate efficacy and unsuitable for use in children less than two years of age. Development of new efficacious vaccines is complicated by the human host-restriction of Salmonella enterica serovar Typhi (S. Typhi) and lack of clear correlates of protection. In this study, we

6 aimed to evaluate the protective efficacy of a single dose of the oral vaccine candidate, M01ZH09, in susceptible volunteers by direct typhoid challenge.we performed a randomised, double-blind, placebocontrolled trial in healthy adult participants at a single centre in Oxford (UK). Participants were allocated to receive one dose of double-blinded M01ZH09 or placebo or 3-doses of open-label Ty21a. Twentyeight days after vaccination, participants were challenged with 104CFU S. Typhi Quailes strain. The efficacy of M01ZH09 compared with placebo (primary outcome) was assessed as the percentage of participants reaching pre-defined endpoints constituting typhoid diagnosis (fever and/or bacteraemia) during the 14 days after challenge. Ninety-nine participants were randomised to receive M01ZH09 (n = 33), placebo (n = 33) or 3-doses of Ty21a (n = 33). After challenge, typhoid was diagnosed in 18/31 (58.1% [95% CI 39.1 to 75.5]) M01ZH09, 20/30 (66.7% [47.2 to 87.2]) placebo, and 13/30 (43.3% [25.5 to 62.6]) Ty21a vaccine recipients. Vaccine efficacy (VE) for one dose of M01ZH09 was 13% [95% CI -29 to 41] and 35% [-5 to 60] for 3-doses of Ty21a. Retrospective multivariable analyses demonstrated that pre-existing anti-vi antibody significantly reduced susceptibility to infection after challenge; a 1 log increase in anti-vi IgG resulting in a 71% decrease in the hazard ratio of typhoid diagnosis ([95% CI 30 to 88%], p = 0.006) during the 14 day challenge period. Limitations to the study included the requirement to limit the challenge period prior to treatment to 2 weeks, the intensity of the study procedures and the high challenge dose used resulting in a stringent model.despite successfully demonstrating the use of a human challenge study to directly evaluate vaccine efficacy, a single-dose M01ZH09 failed to demonstrate significant protection after challenge with virulent Salmonella Typhi in this model. Anti-Vi antibody detected prior to vaccination played a major role in outcome after challenge.clinicaltrials.gov (NCT ) and EudraCT (number ). Feasey NA et al. Rapid emergence of multidrug resistant, H58 lineage Salmonella Typhi in Blantyre, Malawi. PLoS Negl Trop Dis ;9:e Between 1998 and 2010, S. Typhi was an uncommon cause of bloodstream infection (BSI) in Blantyre, Malawi and it was usually susceptible to first-line antimicrobial therapy. In 2011 an increase in a multidrug resistant (MDR) strain was detected through routine bacteriological surveillance conducted at Queen Elizabeth Central Hospital (QECH).Longitudinal trends in culture-confirmed Typhoid admissions at QECH were described between A retrospective review of patient cases notes was conducted, focusing on clinical presentation, prevalence of HIV and case-fatality. Isolates of S. Typhi were sequenced and the phylogeny of Typhoid in Blantyre was reconstructed and placed in a global context.between , there were a mean of 14 microbiological diagnoses of Typhoid/year at QECH, of which 6.8% were MDR. This increased to 67 in 2011 and 782 in 2014 at which time 97% were MDR. The disease predominantly affected children and young adults (median age 11 [IQR 6-21] in 2014). The prevalence of HIV in adult patients was 16.7% [8/48], similar to that of the general population (17.8%). Overall, the case fatality rate was 2.5% (3/94). Complications included anaemia, myocarditis, pneumonia and intestinal perforation. 112 isolates were sequenced and the phylogeny demonstrated the introduction and clonal expansion of the H58 lineage of S. Typhi.Since 2011, there has been a rapid increase in the incidence of multidrug resistant, H58-lineage Typhoid in Blantyre. This is one of a number of reports of the re-emergence of Typhoid in Southern and Eastern Africa. There is an urgent need to understand the reservoirs and transmission of disease and how to arrest this regional increase. Froeschle JE et al. Duration of Vi antibodies in participants vaccinated with Typhim Vi (Typhoid Vi polysaccharide vaccine) in an area not endemic for typhoid fever. Vaccine. 2009;28: After a single injection of Typhim Vi (typhoid Vi polysaccharide vaccine), serum antibody concentrations were monitored for 3 years in 37 adults who resided where typhoid fever was not endemic. Anti-Vi antibody concentrations declined progressively during the study, to levels that support the current US recommendation for revaccination every 2 years.

7 Gaind et al. Molecular characterization of ciprofloxacin-resistant Salmonella enterica serovar Typhi and Paratyphi A causing enteric fever in India. J Antimicrob Chemother. 2006;58: Objectives: To define the genetic characteristics and resistance mechanisms of clinical isolates of Salmonella enterica serovar Typhi (S. Typhi) and S. enterica serovar Paratyphi A (S. Paratyphi A) exhibiting high-level fluoroquinolones resistance. Methods: Three S. Typhi and two S. Paratyphi A ciprofloxacin-resistant isolates (MICs > 4 mg/l) were compared with isolates with reduced susceptibility to ciprofloxacin (MICs mg/l) by PFGE, plasmid analysis, presence of integrons and nucleotide changes in topoisomerase genes. Results: In S. Typhi and Paratyphi A, a single gyra mutation (Ser-83 Phe or Ser-83 Tyr) was associated with reduced susceptibility to ciprofloxacin (MICs mg/l); an additional mutation in parc (Ser-80 Ile, Ser-80 Arg, Asp-69 Glu or Gly-78 Asp) was accompanied by an increase in ciprofloxacin MIC ( 0.5 mg/l). Three mutations conferred ciprofloxacin resistance: two in gyra (Ser-83 Phe and Asp-87 Asn or Asp-87 Gly) and one in parc. This is the first report of parc mutations in S. Typhi. Ciprofloxacin-resistant S. Typhi and S. Paratyphi A differed in their MICs and mutations in gyra and parc. Moreover S. Typhi harboured a 50 kb transferable plasmid carrying a class 1 integron (dfra15/aada1) that confers resistance to co-trimoxazole and tetracycline but not to ciprofloxacin. PFGE revealed undistinguishable XbaI fragment patterns in ciprofloxacin-resistant S. Typhi as well as in S. Paratyphi A isolates and showed that ciprofloxacin-resistant S. Typhi have emerged from a clonally related isolate with reduced susceptibility to ciprofloxacin after sequential acquisition of a second mutation in gyra. Conclusions: To our knowledge this is the first report of molecular characterization of S. Typhi with full resistance to ciprofloxacin. Notably, the presence of a plasmidborne integron in ciprofloxacin-resistant S. Typhi may lead to a situation of untreatable enteric fever. Gilman RH et al. Evaluation of a UDP-glucose-4-epimeraseless mutant of Salmonella typhi as a live oral vaccine. J Infect Dis. 1977;136: A mutant (Ty21a) of Salmonella typhi, which lacks the enzyme uridine 5'-diphosphate-glucose-4- epimerase, was evaluated in volunteers for use as a live attenuated oral typhoid vaccine. Five to eight doses of vaccine (containing 3-10(10) viable organisms per dose) were given to 155 men without significant side effects. The rate of excretion of the vaccine strain in stools was low, and the majority of isolations occurred on day 1 after vaccination. Revertants able to fement galactose were not found in any of 958 stool isolates tested. The mutant, strain Ty21a, grown in brain-heart infusion broth (BHIB) with 0.1% galactose, produces more O side chain than the same vaccine strain cultivated without galactose. Volunteers vaccinated with strain Ty21a grown in galactose and then challenged with 10(5) virulen S. typhi were significantly protected from disease and also had decreased stool carriage of S. typhi as compared with controls. Strain Ty21a grown without galactose did not provide vaccinees significant protection nor decrease fecal excretion of S. typhi as compared with controls. Strain Ty21a, when grown in BHIB with 0.1% galactose, results in a safe, stable and protective oral vaccine that warrants further study in field trials. Gonzalez-Escobedo G et al. Chronic and acute infection of the gall bladder by Salmonella Typhi: understanding the carrier state. Nat Rev Microbiol. 2011;9:9-14. Despite major treatment and prevention efforts, millions of new typhoid infections occur worldwide each year. For a subset of infected individuals, Salmonella enterica subsp. enterica serovar Typhi colonizes the gall bladder and remains there long after symptoms subside, serving as a reservoir for the further spread of the disease. In this Progress article, we explore recent advances in our understanding of the mechanisms by which Salmonella spp. predominantly S. Typhi colonize and persist in the human gall bladder.

8 International Typhoid Consortium et al. Molecular surveillance identifies multiple transmissions of typhoid in West Africa. PLoS Negl Trop Dis. 2016;10:e The burden of typhoid in sub-saharan African (SSA) countries has been difficult to estimate, in part, due to suboptimal laboratory diagnostics. However, surveillance blood cultures at two sites in Nigeria have identified typhoid associated with Salmonella enterica serovar Typhi (S. Typhi) as an important cause of bacteremia in children.a total of 128 S. Typhi isolates from these studies in Nigeria were whole-genome sequenced, and the resulting data was used to place these Nigerian isolates into a worldwide context based on their phylogeny and carriage of molecular determinants of antibiotic resistance.several distinct S. Typhi genotypes were identified in Nigeria that were related to other clusters of S. Typhi isolates from north, west and central regions of Africa. The rapidly expanding S. Typhi clade (H58) previously associated with multiple antimicrobial resistances in Asia and in east, central and southern Africa, was not detected in this study. However, antimicrobial resistance was common amongst the Nigerian isolates and was associated with several plasmids, including the IncHI1 plasmid commonly associated with S. Typhi.These data indicate that typhoid in Nigeria was established through multiple independent introductions into the country, with evidence of regional spread. MDR typhoid appears to be evolving independently of the haplotype H58 found in other typhoid endemic countries. This study highlights an urgent need for routine surveillance to monitor the epidemiology of typhoid and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions to reduce the substantial morbidity and mortality of typhoid. Jin C et al. Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of Salmonella Typhi: a randomised controlled, phase 2b trial. Lancet. 2017;390: Background: Salmonella enterica serovar Typhi ( S Typhi) is responsible for an estimated 20 million infections and deaths each year in resource poor regions of the world. Capsular Vipolysaccharide-protein conjugate vaccines (Vi-conjugate vaccines) are immunogenic and can be used from infancy but there are no efficacy data for the leading candidate vaccine being considered for widespread use. To address this knowledge gap, we assessed the efficacy of a Vi-tetanus toxoid conjugate vaccine using an established human infection model of S Typhi. Methods: In this singlecentre, randomised controlled, phase 2b study, using an established outpatient-based human typhoid infection model, we recruited healthy adult volunteers aged between 18 and 60 years, with no previous history of typhoid vaccination, infection, or prolonged residency in a typhoid-endemic region. Participants were randomly assigned (1:1:1) to receive a single dose of Vi-conjugate (Vi-TT), Vipolysaccharide (Vi-PS), or control meningococcal vaccine with a computer-generated randomisation schedule (block size 6). Investigators and participants were masked to treatment allocation, and an unmasked team of nurses administered the vaccines. Following oral ingestion of S Typhi, participants were assessed with daily blood culture over a 2-week period and diagnosed with typhoid infection when meeting pre-defined criteria. The primary endpoint was the proportion of participants diagnosed with typhoid infection (ie, attack rate), defined as persistent fever of 38 C or higher for 12 h or longer or S Typhi bacteraemia, following oral challenge administered 1 month after Vi-vaccination (Vi-TT or Vi-PS) compared with control vaccination. Analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT , and is ongoing. Findings: Between Aug 18, 2015, and Nov 4, 2016, 112 participants were enrolled and randomly assigned; 34 to the control group, 37 to the Vi-PS group, and 41 to the Vi-TT group. 103 participants completed challenge (31 in the control group, 35 in the Vi-PS group, and 37 in the Vi-TT group) and were included in the per-protocol population. The composite criteria for typhoid diagnosis was met in 24 (77%) of 31 participants in the control group, 13 (35%) of 37 participants in the Vi-TT group, and 13 (35%) of 35 participants in the Vi-PS group to give

9 vaccine efficacies of 54 6% (95% CI ) for Vi-TT and 52 0% ( ) for Vi-PS. Seroconversion was 100% in Vi-TT and 88 6% in Vi-PS participants, with significantly higher geometric mean titres detected 1-month post-vaccination in Vi-TT vaccinees. Four serious adverse events were reported during the conduct of the study, none of which were related to vaccination (one in the Vi-TT group and three in the Vi-PS group). Interpretation : Vi-TT is a highly immunogenic vaccine that significantly reduces typhoid fever cases when assessed using a stringent controlled model of typhoid infection. Vi-TT use has the potential to reduce both the burden of typhoid fever and associated health inequality. Kantele A. Antibody-secreting cells in the evaluation of the immunogenicity of an oral vaccine. Vaccine. 1990;8: The immune response to different dosage schedules of oral live Salmonella typhi Ty21a vaccines was studied by enumeration of specific antibody-secreting cells (ASC) in the peripheral blood believed to have been stimulated by the vaccine antigen on mucosal surfaces and to be on their way back to those sites for local antibody secretion. Four groups of subjects were vaccinated with either three (3 S), two (2 S) or one (1 S) dose of a suspension-formulated vaccine, or with three doses of vaccine in entericcoated capsules (3 E). The ASC-responses were highest in group 3 S, followed by 3 E, 2 S and 1 S, in this order. These differences parallel differences in protection from disease as observed in field trails with these regimens. This assay might therefore be useful for presumptive assessment of the protective ability of new vaccines or vaccine regimens. It certainly can be used to measure the immunogenicity of an oral vaccine. Kariuki S et al. Typhoid in Kenya is associated with a dominant multidrug-resistant salmonella enterica serovar typhi haplotype that is also widespread in Southeast Asia. J Clin Microbiol. 2010;48: In sub-saharan Africa, the burden of typhoid fever, caused by Salmonella enterica serovar Typhi, remains largely unknown, in part because of a lack of blood or bone marrow culture facilities. We characterized a total of 323 S. Typhi isolates from outbreaks in Kenya over the period 1988 to 2008 for antimicrobial susceptibilities and phylogenetic relationships using single-nucleotide polymorphism (SNP) analysis. There was a dramatic increase in the number and percentage of multidrug-resistant (MDR) S. Typhi isolates over the study period. Overall, only 54 (16.7%) S. Typhi isolates were fully sensitive, while the majority, 195 (60.4%), were multiply resistant to most commonly available drugs ampicillin, chloramphenicol, tetracycline, and cotrimoxazole; 74 (22.9%) isolates were resistant to a single antimicrobial, usually ampicillin, cotrimoxazole, or tetracycline. Resistance to these antibiotics was encoded on self-transferrable IncHI1 plasmids of the ST6 sequence type. Of the 94 representative S. Typhi isolates selected for genome-wide haplotype analysis, sensitive isolates fell into several phylogenetically different groups, whereas MDR isolates all belonged to a single haplotype, H58, associated with MDR and decreased ciprofloxacin susceptibility, which is also dominant in many parts of Southeast Asia. Derivatives of the same S. Typhi lineage, H58, are responsible for multidrug resistance in Kenya and parts of Southeast Asia, suggesting intercontinental spread of a single MDR clone. Given the emergence of this aggressive MDR haplotype, careful selection and monitoring of antibiotic usage will be required in Kenya, and potentially other regions of sub-saharan Africa. Keddy KH et al. Fluoroquinolone-resistant typhoid, South Africa. Emerg Infect Dis. 2010;16: No Abstract Available. Keddy KH et al. Persistence of antibodies to Salmonella typhi Vi capsular polysaccharide vaccine in South African school children ten years after immunization. Vaccine. 1999;17:

10 Between 10 and 11 years after children were vaccinated with Vi capsular polysaccharide of Salmonella typhi or meningococcal A+C control vaccine in a double blind randomized trial, we traced 83 subjects, aged years. A blood sample was taken for determination of Vi antibody titres in both groups by radioimmunoassay. TO and TH titres were also done to assess if the participants had had recent exposure to typhoid fever. Fifty-eight percent of subjects in both groups had protective levels of Vi antibody against Salmonella typhi (a titre greater than 1 μg ml 1). There was no significant difference in the levels of Vi antibodies in the cases versus the controls (p=0.5). Two of the children who had received meningococcal A+C vaccine had recently had typhoid fever. Our data show that adolescents in typhoid endemic areas have high levels of Vi antibodies regardless of previous vaccination status, suggesting that Vi antibodies are acquired in adolescence by a large percentage of the population in this area. Moreover, Vi vaccination has led to ongoing antibody production in greater than 50% of Vi vaccinated children in an endemic area for a period of 10 years. Ongoing antigenic exposure may have contributed to these antibody levels. Keitel WA et al. Clinical and serological responses following primary and booster immunization with Salmonella typhi Vi capsular polysaccharide vaccines. Vaccine. 1994;12: Clinical and serum antibody responses following intramuscular injection of two formulations of Salmonella typhi Vi capsular polysaccharide (Vi) were assessed in a double-blind evaluation. Healthy adults were randomly assigned to receive a 25 micrograms dose of liquid (Vi-Liq; n = 182) or freeze-dried Vi vaccine (Vi-Lyoph; n = 55), or placebo (n = 86). Erythema and/or induration > or = 1 cm in diameter at the injection site developed in 13/182 (7%) of Vi-Liq and 3/55 (5%) of Vi-Lyoph recipients (not significant, n.s.). Fever (oral temperature > or = 100 degrees F (37.8 degrees C)) occurred in < 2% of vaccinees. The frequencies of rises of fourfold or greater and of maximal Vi antibody levels were similar in the two vaccine groups. Fourfold or greater rises in serum Vi antibody levels (RIA) developed in 53% of Vi-Lyoph and 60% of Vi-Liq recipients by 1 week (n.s.), and 98 and 93%, respectively, by 1 month (n.s.). The frequencies of adverse reactions and mean Vi antibody levels following booster immunization with Vi- Liq 27 to 34 months after primary immunization (n = 55) were similar to those observed following primary immunization, although subjects given a booster dose were more likely to develop local reactions > or = 1 cm in diameter than those given a first dose (10/55 versus 13/182, p = by the chi 2 test). Primary and booster immunizations with the Vi vaccines are well tolerated in healthy adults; mean Vi antibody levels remain significantly elevated for up to 34 months after primary immunization. Khan MI et al. Effectiveness of Vi capsular polysaccharide typhoid vaccine among children: a cluster randomized trial in Karachi, Pakistan. Vaccine. 2012;30: Abstract BACKGROUND: Typhoid fever is endemic in Karachi, with an incidence among children ranging from 170 to 450 per 100,000 child-years. Vaccination strategies are important for prevention, and the Vi capsular polysaccharide (ViCPS) vaccine has been shown to be effective in reducing the burden of typhoid fever. METHODS: A cluster randomized trial was conducted in three low socioeconomic urban squatter settlements in Karachi, Pakistan between 2002 and Subsamples were followed up for assessment of immune response and adverse events after vaccination. RESULTS: The study participants were similar in a wide variety of socio-demographic and economic characteristics at baseline. A total of 27,231 individuals of the total target population of 51,965 in 120 clusters either

11 received a ViCPS vaccine (13,238 [52% coverage]) or the control Hepatitis A vaccine (13,993 [53%]). Typhoid fever was diagnosed in 30 ViCPS vaccine recipients and 49 Hepatitis A vaccine recipients with an adjusted total protective effectiveness of 31% (95%CI: -28%, 63%). The adjusted total vaccine protective effectiveness was -38% (95%CI: -192%, 35%) for children aged 2-5 years and 57% (95%CI: 6%, 81%) for children 5-16 years old. CONCLUSION: The ViCPS vaccine did not confer statistically significant protection to children in the study areas, and there was a decline in antibody response 2 years post-vaccination. However, the ViCPS vaccine showed significant total protection in children 5-16 years of age, which is consistent with other studies of ViCPS vaccine conducted in India, Nepal, China and South Africa. These findings suggest that ViCPS vaccination of school-aged children will protect the children of urban, typhoid endemic areas against typhoid fever. Klugman K et al. Protective activity of Vi polysaccharide vaccine against typhoid fever. Lancet. 1987;2: The protective efficacy against typhoid fever of a single intramuscular injection of 25 micrograms of the Vi capsular polysaccharide (CPS) was assessed in a randomised double-blind controlled trial. Vaccination of 11,384 children was followed by 21 months' surveillance. 47 blood-culture-proven cases of typhoid occurred in children who received meningococcal A + C CPS vaccine and 19 cases in those vaccinated with Vi CPS. Protective efficacy was 60% calculated from the day of vaccination and 64% from 6 weeks after vaccination. Surveillance also included 11,691 unvaccinated children; 173 cases occurred in this group. Protective efficacy in relation to the unvaccinated group was 77.4% and 81.0% after 21 months, calculated immediately and 6 weeks after vaccination, respectively. Vaccination was associated with minimum local side-effects, and an increase in anti-vi antibodies occurred, as measured by radioimmunoassay and enzyme-linked immunosorbent assay. Antibody levels remained significantly raised at 6 and 12 months post vaccination. Vi CPS is thus a safe and effective means of typhoid vaccination. Koshiol J et al. Salmonella enterica serovar Typhi and gallbladder cancer: a case control study and meta-analysis. Cancer Med. 2016;5: In Chile, where gallbladder cancer (GBC) rates are high and typhoid fever was endemic until the 1990s, we evaluated the association between Salmonella enterica serovar Typhi (S. Typhi) antibodies and GBC. We tested 39 GBC cases, 40 gallstone controls, and 39 population-based controls for S. Typhi Vi antibodies and performed culture and quantitative polymerase chain reaction for the subset with bile, gallstone, tissue, and stool samples available. We calculated gender and education-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the association with GBC. We also conducted a metaanalysis of >1000 GBC cases by combining our results with previous studies. GBC cases were more likely to have high Vi antibody titer levels than combined controls (OR: 4.0, 95% CI: ), although S. Typhi was not recovered from bile, gallstone, tissue, or stool samples. In our meta-analysis, the summary relative risk was 4.6 (95% CI: , Pheterogeneity=0.6) for anti-vi and 5.0 (95% CI: , Pheterogeneity = 0.2) for bile or stool culture. Our results are consistent with the meta-analysis. Despite differences in study methods (e.g., S. Typhi detection assay), most studies found a positive association between S. Typhi and GBC. However, the mechanism underlying this association requires further investigation Kroon FP et al. Impaired antibody response after immunization of HIV-infected individuals with the polysaccharide vaccine against Salmonella typhi (Typhim-Vi). Vaccine. 1999;17: Infections with Salmonella species, including Salmonella typhi, are more frequently observed in HIVinfected individuals than in healthy individuals. HIV-infected individuals were vaccinated with

12 polysaccharide vaccine against Salmonella typhi (Typhim-Vi ) which is assumed to be a T-cellindependent antigen. We found that the antibody response in patients with < /l CD4+ T lymphocytes was significantly lower compared with patients with /l CD4+ T lymphocytes and healthy controls. The antibody response after vaccination with the polysaccharide salmonella Viantigen was correlated with the number of CD4+ T lymphocytes and therefore Typhim-Vi can be considered to be a T-cell-independent type 2 antigen. The results of this study indicate that after vaccination the proportion of HIV-infected individuals with protective antibody concentrations against Salmonella typhi will be lower than in healthy controls. Levine MM. Chapter 61: Typhoid fever vaccines. In: Plotkin s Vaccines, 2017;Seventh Edition: No abstract available. Levine MM et al. Duration of efficacy of Ty21a, attenuated salmonella typhi live oral vaccine. Vaccine. 1999;17:S22-S27. Currently, two different formulations of Ty21a live oral typhoid vaccine are commercialized. The entericcoated capsule formulation was licensed based on results of three years of follow-up of a randomized, placebo-controlled, double-blind field trial in Area Occidente, Santiago, Chile, which demonstrated that three doses of this formulation, given on an every other day immunization schedule, conferred the best protection among several options evaluated. Subsequently, a liquid formulation (lyophilized vaccine organisms reconstituted with buffer and water into a vaccine cocktail) was commercialized after it was shown to provide superior protection than enteric-coated capsules over three years of follow-up in a randomized, placebo-controlled field trial in Area Sur Oriente and Area Norte, Santiago. Surveillance in the Area Occidente trial was continued for four additional years (i.e., total seven years of follow-up) and in the Area Sur Oriente/Area Norte trial for two additional years (i.e., a total of five years of follow-up). These additional surveillance data, which were analyzed to ascertain the longevity of protection conferred by these formulations of Ty21a, revealed that three doses of Ty21a in enteric-coated capsules (every other day schedule) conferred 67% protection over three years and 62% protection over seven years of follow-up, whereas three doses of liquid formulation (every other day schedule) elicited 77% protection over three years and 78% over five years of follow-up. Based on its excellent clinical acceptability, ease of oral administration, proven practicality in school-based mass immunization, and long-term efficacy enduring at least seven years, it is proposed that school-based immunization with Ty21a be utilized as a control measure in areas where the incidence of typhoid fever is high and Salmonella typhi are antibiotic-resistant. Levine MM et al. Large-scale field trial of Ty21a live oral typhoid vaccine in enteric-coated capsule formulation. Lancet. 1987;1: Three doses, given within one week, of Ty21a attenuated Salmonella typhi oral vaccine in an entericcoated formulation provided 67% efficacy for at least 3 years in a randomised, placebo-controlled field trial involving 109,000 schoolchildren in Santiago, Chile. Increasing the interval between doses to twenty-one days did not enhance protection. Significantly less protection followed administration of vaccine in gelatin capsules with sodium bicarbonate. Ty21a provides the same level of protection as the heat/phenol-inactivated whole cell parenteral vaccine but differs in not causing adverse reactions. Ty21a may now be regarded as a practical public health tool. Levine MM et al. Progress in vaccines against typhoid fever. Rev Infect Dis 1989;11:S552-S567. The widely available heat-phenol-inactivated whole cell typhoid vaccine, which provides approximately 65% protection, has limited usefulness because of the adverse reactions it evokes. In contrast, several new